Heroin is a highly addictive drug and its abuse continues to be a major public health problem. Heroin abuse is associated with high mortality due to overdose, high risk of contracting diseases such as HIV/AIDS and viral hepatitis, and crime that often exceed that of most other abused drugs. Extensive research efforts have been dedicated to understanding the regulation of 5-opioid receptor (MOR), molecular target of heroin metabolites such as morphine, but limited studies have been conducted directly in the human brain regarding MOR and its intracellular signaling in relation to heroin abuse. Heroin abuse has a high genetic load and mutation of the MOR gene (OPMR1) has been linked with heroin abuse risk. During the first funding period for our studies, we documented that H 90% of subjects with the A118G mutation in our postmortem brain bank collection, from a homogenous Caucasian population, were heroin abusers. Neurobiological studies revealed that the polymorphism was relevant to opioid neuropeptide expression in the nucleus accumbens linked to reward and goal-directed behavior. Moreover, subjects with the 118G allele had higher functional coupling of the MOR in the nucleus accumbens. The proenkephalin (PENK) opioid neuropeptide is also strongly associated with reward and individuals with genetic polymorphism of this gene also had sensitized opioid receptor G-protein coupling in the nucleus accumbens. It is the aim of the next phase of our project to investigate more thoroughly the regulation of MOR; the intracellular G-protein signaling cascades that mediates the physiological actions of this receptor in heroin abusers and in relation to the OPMR1 polymorphism. Factors that modulate MOR function such as receptor density, phoshorylation state, dimerization with 4-opioid receptors, and coupling to 2-arrestin will be studied. Cluster and network analysis will be conducted on RNA data already collected from microarray analysis and customized real-time PCR datasets to identify the intracellular regulatory pathways linked to opioid receptor G-protein signaling. Key proteins within these networks will also be studied to validate the functional disturbances in relation to heroin use and the OPMR1 mutation. Expanding knowledge regarding MOR and its intracellular signaling machinery directly in the human brain will provide a major insight as to the neurobiological correlates linked with heroin abuse vulnerability and targets for medication development.